A sudden increase in presynaptic calcium concentration triggered by the arrival of an action potential has been postulated, but never measured, to be the key requirement for vesicle mobilization in synaptic transmission. I propose to implement a combination of optical and electrophysiological techniques to measure fast intracellular calcium concentration changes elicited by action potentials in a vertebrate neuromuscular synaptic co- culture preparation. Specifically, I will use a confocal spot detection methodology, which provides a temporal resolution of tens of microseconds and a spatial resolution of sub microns to investigate the role of presynaptic calcium and calcium activated potassium conductances in the generation of intracellular calcium transients in whole cell voltage clamped presynaptic varicosities. In addition, flash photolysis of the caged calcium compound DM- nitrophen will be used to study the independent role of calcium concentration changes in releasing neurotransmitter. Simultaneous measurements of evoked postsynaptic currents and calcium transients will provide quantitative data to test current models correlating presynaptic calcium concentrations with neurotransmitter release at the neuromuscular junction. The proposed experiments will help elucidate the mechanisms by which presynaptic conductances at active zones, when opened by action potentials, may generate gradients in the presynaptic calcium concentration that ultimately modulate neurotransmitter release.